Previous reports from our laboratory have found that nerve growth factor differentiated pheochromacytoma[unreadable] cell lines (NGFDPC12 cells) exposed to pathological levels of exogenous palmitic (PA) and stearic (SA) fatty[unreadable] acids -- at a level comparable to that found during hypoxia/ischemia - causes a dramatic decrease in cell[unreadable] viability and apoptosis. This decrease in cell survival occurs because these saturated FFA induced massive[unreadable] apoptosis (program cell death) during 24 hours exposure. In contrast, similar levels of arachidonic (AA) or[unreadable] oleic (OL), EPA or DMA FFA did not decrease the viability of these cells. The central hypothesis states that[unreadable] high levels of PA or SA fatty acids induce neuronal apoptosis secondary to mitochondria! dysfunction. This[unreadable] application will primarily focus on characterizing the apoptotic effect of PA and SA in NGFDPC12 cells and[unreadable] extend this analysis to primary retinal ganglion and cortical cell neurons. Specific Aim 1 will include the[unreadable] characterization of PA and SA-mediated caspase-independent neuronal cell death. This aim will determine[unreadable] the effects of these saturated FFA in the survival of rat cortical and retinal ganglion primary cells in culture.[unreadable] The experimental design will evaluate whether the loss of cell viability is result of apoptosis or necrosis and[unreadable] will determine whether the process is dependent on caspase activation. Further experiments will analyze[unreadable] what specific apoptotic proteins are involved in the process. Specific Aim 2 will determine whether FFAmediated[unreadable] neuronal cell death occurs through the mitochondrial pathway. Preliminary cDNA array analysis[unreadable] and Quantitative real time PCR data has identified several candidate mitochondrial genes that are involved[unreadable] in this process. This aim will elucidate the role of these genes by using siRNA knockdown and[unreadable] overexpression to modulate cellular levels. We anticipate that these studies will help us to clarify the[unreadable] mechanisms of how pathological levels of saturated FFA affect neuronal survival and apoptosis.